Localization of Aromatase in Synaptosomal and Microsomal Subfractions of Quail (Coturnix coturnix japonica) Brain

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Abstract
The subcellular distribution patterns of aromatase, 5α- and 5β-reductase in the hypothalamus/preoptic area of Japanese quail were studied using standard methods of centrifugation, and fractional constituents were identified by marker enzymes. Aromatase was concentrated 8-fold in the 100,000 g pellet (P3) along with a 3-fold enrichment in the microsomal marker NADPH-cytochrome c reductase (NCR) a result consistent with glandular tissues. In addition, aromatase was enriched 2-fold in the 11,000 g pellet (P2) and, owing to its large size, this fraction accounted for more total activity than P3. Although P2 contained the mitochondrial marker succinate dehydrogenase (SDH), treatment with Triton X-100 to solubilize membranes and release occluded enzymes increased measured NCR and the cytosol marker lactate dehydrogenase (LDH) 2- and 4-fold, respectively – evidence that this fraction was composed of mitochondria plus synaptosomes (pinched-off nerve terminals). To further explore the location of aromatase in the 11,000 g fraction, P2 was exposed to hypotonic buffer, a treatment known to cause lysis of synaptosomes, and then separated into three fractions P2a (11,000 g pellet), P2b (100,000 g pellet) and P2s (100,000 g supernatant). Aromatase colocalized with the microsomal marker NCR (13- and 4-fold increase, respectively) in the 100,000 g (P2b) pellet which was, however, devoid of mitochondrial enzyme activity. We infer from this that a significant portion of aromatase in brain is associated with smooth membranes present inside synaptosomes. 5β-Reductase in quail brain subfractions was enriched 6-fold in the 100,000 g supernatant together with a 4-fold enrichment in the cytosolic marker LDH. A smaller amount of 5β-reductase activity was associated with the P2 (synaptosomal) pellet, and activity increased 8-fold in the supernatant derived therefrom. A corresponding increase in the cytosolic marker LDH (11-fold) is evidence that these enzymes, like aromatase, are contained within synaptosomes. The specific activity of 5α-reductase was not enriched in any one subfraction; however, the majority of the cellular activity (64%) was associated with the 1,000 g pellet, a heterogeneous fraction including cell nuclei. When immature quail were exposed to long days (16:8 h light:dark), a treatment known to induce testicular maturation and to increase aromatase in brain homogenates, microsomal enzyme activity increased 2-fold whereas synaptosomal activity increased 6-fold; 5α-and 5β-reductase activities were unchanged. These experiments provide additional evidence for a dual subcellular localization of aromatase and suggest that activity may be differentially regulated in the two cellular compartments. Based on data reported here, we conclude that nerve terminals are a previously unrecognized site of aromatization in brain and suggest that estrogens synthesized at the synapse, in contrast to that taken up from the circulation, may have unique cellular targets and mechanisms of action.